The developmental and tissue-specific control o hemoglobin synthesis is a complex process involving two switches which control the expression of specific hemoglobin genes; for example, the beta-like globin genes switch from embryonic (epsilon) to fetal (gamma) to adult (beta). A number of trans-acting proteins and cis-acting DNA elements have been found to be part of the switching machinery, including the silencer located in the region between -250 and -300 bp 5' to the epsilon-globin gene promoter, which was first discovered in this laboratory. Further characterization of the silencer will provide not only answers to basic questions regarding the regulation of hemoglobin gene expression, but also better understanding of the problems related to hemoglobinopathies. In order to further characterize the DNA sequences functioning co-operatively with the silencer, we have constructed a plasmid, pE6000, which contains the epsilon-globin gene promoter, its 5' upstream flanking sequences up to 6021 bpo and a reporter gene. This construct has been deleted progressively from he far 5' end to give a series of nested deletion mutants with various lengths of the upstream sequence. Some of these constructs have been transiently transfected into K562, a human erythroleukemia cell line and different levels of expression of the reporter gene, luciferase gene, were observed with different 5' upstream deletions. In transient transfection assays, deletion of 5' sequences between -935 and -5824 bp and between -2832 and =5824 bp changes the epsilon-globin gene promoter activity. This result indicates that DNA sequences located in the region between -900 and -6000 bp may have multiple control elements to regulate epsilon-globin gene expression.